The objective of the proposed research is to improve exposure assessment of pesticides by developing a methodology for measuring acetylcholinesterase (AChE) inhibition that includes determinations of free and total enzyme in the same blood sample. The research will develop a methodology for preparing whole blood samples so that these measurements can be made sequentially using currently available methods for measuring cholinesterase activity that are used in clinical monitoring of pesticide exposure. The research proposes to calibrate colorimetric measurements of these AChE activities with specific binding of a radiolabeled probe to AChE in blood samples partially inhibited by an organophosphate (OP) pesticide. Once analyzed by standard AChE assays, the samples will be processed to reactivate the OP-inhibited AChE followed by determination of this reactivated fraction through binding of the AChE- specific probe and colorimetric assay. This parallel AChE measurement approach is intended to validate the sequential colorimetric determinations so that the methodology can be performed without the radioactive assay. This capability will improve exposure assessment of pesticides in several ways. First, knowledge of the total AChE activity in a blood sample will permit a more accurate estimate of the level of inhibition by utilizing individual- specific information. Secondly, measurement of AChE activity is considered to be a more relevant endpoint for the neurological effects of pesticides than other cholinesterase activities. Finally, the ratio of inhibited-to-total AChE will provide an internal standard to normalize the different ChE assays currently used to monitor pesticide exposure.